Biochip is a device that can analyze genetic information and protein information automatically in a large scale, or detect the presence and the function of a biomolecule easily and rapidly. This biochip is being actively applied for various fields including gene and protein researches, medicines, and agricultural, environmental and chemical industries, etc.
The biochip is classified broadly to genotyping chip, expression chip and microfluidics chip: the genotyping DNA chip is to detect the presence of a particular gene by using a probe; the expression DNA chip is to monitor the expression profiling of gene associated with a particular disease; and the microfluidics chip is to detect the presence and/or the reaction of a biomolecule within a sample including blood and urine. Presently, the genotyping DNA chip is commercialized and used widely in research areas and medical diagnosis areas.
Generally, the term microarray chip defines a chip that arrays hundreds to ten thousands kinds of genes or proteins mounting on a glass plate by using a microarray apparatus. Among these, DNA chip is to microarray oligonucleotides as probes on a glass plate in order to identify the presence of a particular gene with a fluorescence scanner.
The DNA chip is being utilized practically for research and diagnosis fields. Particularly, this chip is applied to elucidate the gene function including cellular metabolism, physiological phenomena and mutual relation between genes by using gene expression profiling and genotyping techniques, etc. The DNA chip is also used widely in diagnosis to examine a mechanism causing a particular disease such as cancer, prognostic diagnosis and action of drugs, to identify genetic information of microbes causing diseases, and to screen mutations, etc.
Diagnostic DNA chip has been developed in 1994 by Dr. Steve Fodor in Affymetrix Co. Ltd., and the first HIV gene chip started to be commercialized in a market. Nowadays, researches upon the diagnostic DNA chip are attempted actively in order to diagnose chronic diseases including HIV, rheumatism, autoimmune disease, chronic nephritis, atherosclerosis, atopic dermatitis and allergy, etc. Especially, recent studies upon DNA chips tend to develop chips for diagnostic use rather than chips for research use. Moreover contrasting to genotyping chips useful for diagnosing genotype, pathogen and virus, an approach on gene expression profiling capable of diagnosing various diseases including cancer and leukemia, is being accomplished.
Recently, microfluidics chip (Lab-on-a chip) that can detect a lot of diseases coincidentally from one trial and predict outbreak of diseases from genetic information of an individual by introducing IT and nano technologies, attracts attention. The microfluidics chip is also referred to as biochip. This chip is used to analyze a reaction profiling of various biomolecules within a chip, after a minute amount of an analytic target material (DNA, RNA, peptide, protein, etc.) is introduced into a chip chamber. This biochip is to detect the presence and/or the reaction of a biomolecule by monitoring changes of electrical property from an electrode installed in a chip, after being reacted with the biomolecule in a reaction chamber.
Such a biochip is highly applicable for medical diagnosis, because it can identify the presence and/or the reaction of a biomolecule more easily and rapidly by detecting electrical signals than any other DNA chips mentioned above.
For example, HPV DNA chip is a device that prepares HPV oligonucleotide probes and microarrays these probes on a glass plate in order to diagnose whether HPV, a pathogenic virus causing cervical cancer is positive or not. Nevertheless, it is impossible to directly diagnose the positive status of HPV, right after suspected sample is collected. That is to say, primers for amplifying HPV viral gene, labeled with fluorescence should be prepared in advance. Then, the collected sample should be amplified by performing a PCR, mounted onto an HPV DNA chip and monitored to examine a fluorescent signal with a fluorescence scanner. However, this system for detecting hybrids by using a DNA chip (laser-induced fluorescence) is inconvenient to be manipulated and spends time a lot. Therefore, this method has various problems and disadvantages. It needs high cost due to labeling a DNA sample with a fluorescent material and is not portable because of using an expensive fluorescence scanner.
In contrast, the biochip can identify the presence and/or the reaction of a biomolecule relatively easily and rapidly by detecting electrical signals. Particularly, the biochip can detect the presence and/or the reaction of a biomolecule (for example, DNA) by using electrical signals rather than fluorescent signals. More particularly, the biochip adopts a system for detecting changes electrically, in which the change of an impedance value (or a capacitance value) is monitored after reacting a receptor immobilized onto an electrode with a biomolecule, or the change of an impedance value (or a capacitance value) is monitored after reacting between biomolecules in a chip chamber.
For example, it is reported in the PCR process that dNTP should degrade to dNMP and diphosphate and the resulting dNMP is polymerized simultaneously from a primer complementary to a DNA template sequence so as to synthesize DNA. Accordingly, the impedance value within a PCR reagent increases as DNA concentration increases (See Korean Patent Laid-open NO. 10-2004-0042021). Therefore, it is possible to determine whether the PCR reaction is performed and a particular DNA sequence exists or not, when a PCR reaction chamber is manufactured with a biochip structure and the changes of an impedance value in a reagent are detected electrically on an electrode installed in such a biochip.
In addition, it is possible to detect electrically whether a PCR reaction or a hybridization reaction is accomplished and/or whether a target nucleotide sequence exists or not, when oligonucleotides such as primers or probes are immobilized on a biochip electrode, a PCR reaction or a hybridization reaction is conducted and then, the changes of a capacitance or impedance value are measured with the electrode on which the oligonucleotides are immobilized.
In general, the biochip adopts a system for determining the reaction and the presence of a particular biomolecule by monitoring the impedance change with an electrode equipped in the biochip after reacting the biomolecule and other reagents (for example, the receptor immobilized onto an electrode). Accordingly in order to guarantee the reliability of a biochip, it is important to measure the change of the impedance value accurately. Generally, impedance (Z) indicates the sum of resistance (R) as a real number portion and reactance (X) as an imaginary number portion (See following Mathematical formula 1), and the magnitude of impedance corresponds to a square root of resistance score (R) and reactance score (X) (See following Mathematical formula 2).
                                                        Z              =                              R                +                                  j                  ⁢                                                                          ⁢                  X                                                                                                        =                              R                +                                  j                  ⁡                                      (                                                                  X                        L                                            -                                              X                        C                                                              )                                                                                                                          =                              R                +                                                      j                    ⁡                                          (                                                                        ω                          ⁢                                                                                                          ⁢                          L                                                -                                                  1                                                      ω                            ⁢                                                                                                                  ⁢                            C                                                                                              )                                                        ⁡                                      [                    Ω                    ]                                                                                                          [                  Mathematical          ⁢                                          ⁢          formula          ⁢                                          ⁢          1                ]                                                                                                    Z                                            =                                                                    R                    2                                    +                                      X                    2                                                                                                                          =                                                                                          R                      2                                        +                                                                  (                                                                              ω                            ⁢                                                                                                                  ⁢                            L                                                    -                                                      1                                                          ω                              ⁢                                                                                                                          ⁢                              C                                                                                                      )                                            2                                                                      ⁡                                  [                  Ω                  ]                                                                                        [                  Mathematical          ⁢                                          ⁢          formula          ⁢                                          ⁢          2                ]            
Accordingly, the impedance (Z) value is made to have a correlation with reactance (X). Also, the reactance (X) has a correlation with the capacitance (C) value because it is ωL−1/ωC. Therefore, the change of the capacitance (C) value varying according to biological, biochemical or chemical reactions, is reflected by the change of the impedance value, which enables to check the reaction and/or the presence of a biomolecule after its measurement. Finally, it is verified that the change of the capacitance value should change the impedance value in a biochip and influence upon the sensitivity of the biochip.
The change of the impedance value can be affected by two kinds of capacitance values after reacting the biomolecule and other reagents (for example, the receptor immobilized onto an electrode). Particularly, when the change of the capacitance value is measured by the electrode of a biochip, total change of capacitance value (CT) in a biochip can include change of a capacitance value (Cd) on the surface of a sensing electrode after reacting a receptor immobilized onto an electrode with a biomolecule, and a capacitance value (Ct) between an imaginary electrode plate and another sensing electrode, which is different from the change of the capacitance value (Cd) (See following Mathematical formula 3).
                              C          T                =                  1                                    1                              C                t                                      +                          1                              C                d                                                                        [                  Mathematical          ⁢                                          ⁢          formula          ⁢                                          ⁢          3                ]            
In a biochip, the biomolecule is reacted under a reagent filling a reaction chamber and the reagent generally contains a buffer solution or electrolytes. The buffer solution and electrolytes contain ions to influence conductance between sensing electrodes of a biochip. This buffer effect upon the conductance between sensing electrodes further affects the capacitance value (Ct) between the imaginary electrode plate and another sensing electrode, which are contained in the buffer functioning as a dielectric substance. Finally, this influences total change of the capacitance value (CT).
On the other hand, the capacitance value depends on the dielectric constant of a material between electrodes as defined in following Mathematical formula 4. It is verified that when the dielectric constant of a material between electrodes is small, the capacitance value becomes small.
                                                        C              =                            ⁢                                                Q                  V                                =                                  ɛ                  ⁢                                      A                    t                                                                                                                                        ⁢                              A                ⁢                                  :                                ⁢                                                                  ⁢                area                ⁢                                                                  ⁢                of                ⁢                                                                  ⁢                electrode                                                                                                      ⁢                              T                ⁢                                  :                                ⁢                                                                  ⁢                interval                ⁢                                                                  ⁢                between                ⁢                                                                  ⁢                electrodes                                                                                                      ⁢                              ɛ                ⁢                                  :                                ⁢                                                                  ⁢                dielectric                ⁢                                                                  ⁢                constant                ⁢                                                                  ⁢                of                ⁢                                                                  ⁢                material                            ⁢                                                                                                                                    ⁢                              between                ⁢                                                                  ⁢                electrodes                                                                        [                  Mathematical          ⁢                                          ⁢          formula          ⁢                                          ⁢          4                ]            
However, the reagent such as the above-mentioned buffer has a small dielectric constant due to the ion conductivity and thus, 1/Ct, an element comprising total change of the capacitance value (CT) becomes large in Mathematical formula 3. Accordingly, when the biomolecule is reacted under a buffer solution filled in the reaction chamber of a biochip, the capacitance value (Cd) changed by the reaction between the sensing electrode and the biomolecule can be absorbed into the capacitance value (Ct) between the imaginary electrode plate and the another sensing electrode contained in the buffer functioning as a dielectric substance. Particularly as demonstrated in the Mathematical formula 3, the capacitance value (Cd) changed on the surface of the sensing electrode by reacting the receptor immobilized onto the electrode with the biomolecule does not influence the total change of the capacitance value (CT), because the reciprocal number (1/Ct) of the capacitance value (Ct) between the imaginary electrode plate and the another sensing electrode filled with the reagent containing buffer ions as a dielectric substance is large. Therefore, it is difficult problematically to determine the presence and/or the reaction of a biomolecule by the electrical detection using the sensing electrode of a biochip.
Referring to this, FIG. 1 illustrates that the curve of the impedance value varied according to frequency is observed to have the same trend before and after PCR reaction, even though a particular target DNA template as a diagnostic sample exists. If conventional biochip could measure PCR reaction using a particular target DNA template as a biomolecule accurately, the change of the capacitance value (Cd) on the surface of a sensing electrode could be detected electrically after the PCR reaction between a primer fixed onto the sensing electrode and a template ssDNA. The change pattern of impedance curves might also be observed before and after the PCR reaction, because the change of the capacitance value (Cd) on the surface of the sensing electrode should be reflected after DNA amplification by the PCR reaction.
However, since the capacitance value (Cd) changed after reacting a sensing electrode and a biomolecule may be passed over by the capacitance value (Ct) between an imaginary electrode plate and another sensing electrode as described above, the impedance curve is not changed and just observed as a single curve before and after the PCR reaction, when being measured with an biochip-connected apparatus monitoring an impedance value (See FIG. 1). Therefore, in biochips of prior arts, the capacitance value (Cd) changed by reacting a receptor immobilized onto an electrode and a biomolecule, is passed over by the capacitance value (Ct) between an imaginary electrode plate and another sensing electrode containing a buffer solution as a dielectric substance, and thus, two curves of the changes of the impedance value which mean total change of the capacitance value (CT) are not measured. Problematically, it is difficult to monitor whether the biomolecule reacts and/or exists or not in practice.
Particularly, in US Patent Laid-open No. 2004/0110277, the system for sensing a hybridization reaction, in which a probe DNA is immobilized onto an electrode of sensor cell in a biosensor and hybridized with a target DNA to change a capacitance value and finally, a current value of a transistor, has been disclosed. However, it does not focus to settle following problem: since the capacitance value (Cd) changed by reacting a receptor immobilized onto an electrode with a biomolecule is passed over by the capacitance value (Ct) of the buffer solution, the reaction between the receptor immobilized onto the electrode and the biomolecule as a target material cannot be detected electrically.
Besides, in US Patent Laid-open NO. 2006/0226030, the technique for sensing a hybridization reaction by detecting a capacitance value with a sensor comprising a plate, an electrode and a catcher molecule immobilized on the plate, in which the catcher molecule is hybridized with DNA single strand labeled with metallic ball (having a different dielectric constant from that of the other material) and as a result, the label surrounds the electrode to change a part of the capacitance value regarding an electrode impedance value, has been disclosed. However, it did not pay attention to overcome following problem: since the capacitance value (Cd) changed by reacting a receptor immobilized onto an electrode with a biomolecule is passed over by the capacitance value (Ct) of a buffer solution, changes of electrical property can not be detected sensitively during actual reaction.
Furthermore, in conventional techniques, a system for increasing sensing sensitivity of IDE by reducing the interval of sensor electrodes in order to improve the sensing sensitivity of a biochip, has been disclosed. But, it did not recognize the problem mentioned above: since the capacitance value (Cd) changed by actual reaction is passed over by the capacitance value (Ct) of a buffer solution, the changes of the capacitance value and the impedance value cannot be detected sensitively.
Therefore, conventional biochips and methods using the same for identifying the reaction and/or the presence of biomolecules disclosed in prior arts should be improved.
In order to settle above-mentioned problems, the present inventors have accomplished to design a method for detecting a biomolecule and a biochip therefor, wherein the dielectric constant of a material filled between electrodes within a reaction chamber of a biochip is made large, which does not distort actual change of the capacitance value when reacting on the surface of a sensing electrode, and thus, reflects accurately the biological, biochemical or chemical reaction of a biomolecule in a reaction chamber.